The MRN-ATM pathway is primarily involved in sensing, signaling, and repairing DNA double-strand breaks (DSBs). DSBs arise as a consequence of external insults, such as ionizing radiation and are generated in all cycling cells during DNA replication. If unrepaired, they may lead to mutations and chromosome rearrangements, translocations or loss. The long-term objective of this proposal is to understand how the MRN-ATM pathway regulates DNA damage response to DSBs. Specifically, we will study how ATM and MRN interact with DNA, how ATM modulates MRN-DNA interactions and conversely how MRN affects ATM's behavior on DNA. Next, we will investigate the role of damaged DNA in ATM activation. Finally, we will determine how MRN and ATM cooperate to maintain genome stability in the presence of DSBs. Our system to analyze these questions remains Xenopus egg cell free extracts. The MRN-ATM pathway is a central node in the response to DSBs and defects in this pathway or its downstream targets are associated with inherited cancer susceptibility syndromes including Ataxia-Telangiectasia (A-T), Ataxia Telangiectasia like disorder (ATLD), Nijmegen Breakage Syndrome (NBS), Fanconi Anemia (FA), BRCA1-associated breast and ovarian cancer susceptibility. We anticipate that our studies will help explain how maintenance of genome stability by the MRN-ATM pathway protects from tumorigenesis. Furthermore, since the MRN-ATM pathway is a target for therapeutic intervention, a better mechanistic understanding of how the MRN-ATM pathway functions could help to identity compounds with radio-protective or radio-sensitizing potential.